A widely used means of genetic regulation in bacteria is a non-protein coding RNA element called a riboswitch. These are cis-acting elements found in the leader sequence of mRNAs and regulate gene expression by directly binding small molecule metabolites to a highly structured receptor domain. This receptor directs folding of a secondary structural switch in a downstream regulatory domain that in turn interfaces with the expression machinery (either RNA polymerase or the ribosome). In a broad spectrum of bacteria, particularly Firmicutes and Fusobacteria, central metabolic pathways including purine, amino acid, and cofactor biosynthesis and transport are regulated by riboswitches. Furthermore, genes essential for survival or virulence are under riboswitch control in a number of medically important pathogens including Listeria monocytogenes, Staphylococcus aureus, Pseudomonas aeruginosa, and Mycobacterium tuberculosis making them of great interest as novel targets for designing antimicrobial therapeutics. In addition, riboswitches are increasingly serving as powerful model systems for developing the tools and methodologies for the design of small molecules that target other RNAs of medical interest. Towards the long-term goal of developing a molecular understanding of how RNA interacts with small molecules and the mechanisms it uses to regulate gene expression, we are using S-adenosylmethionine (SAM)-binding riboswitches as a model system. This proposal details a set of interconnected specific aims that addresses fundamental questions related to these research goals: (1) what is the range of structural diversity across SAM-responsive riboswitches, (2) what is the nature of the unbound structure of SAM-I superfamily riboswitches, (3) which structural features of the aptamer and expression domains play functional roles in regulation, and (4) do binding thermodynamics or kinetics dictate the regulatory response? To address these questions, a combination of approaches including X-ray crystallography, small-angle X-ray scattering (SAXS), and various biochemical and molecular biological approaches will be utilized in a set of experiments specifically designed to study the structure/function linkage. A deeper knowledge of how RNA specifically interacts with small molecules will help pave the way for a new generation of therapeutics that target non-protein coding RNAs that are pervasive in both bacteria and eukaryotes.